Method for enhancing functional properties of submucosal tissue graft constructs

ABSTRACT

A tissue graft construct comprising submucosal tissue of a warm-blooded vertebrate and a proliferating population of cells is described. The submucosal graft constructs used in accordance with the present invention induce host tissue proliferation, remodeling and regeneration of appropriate tissue structures upon implantation in a host.

This is a continuation of Ser. No. 08/386,452, filed Feb. 10, 1995, andnow U.S. Pat. No. 5,695,998.

FIELD OF THE INVENTION

The present invention relates to the culturing of eukaryotic cells. Moreparticularly, this invention is directed to a method for supporting thegrowth and tissue differentiation of eukaryotic cells in vitro, bycontacting the eukaryotic cells with submucosal tissue of a warm-bloodedvertebrate, under conditions conducive to eukaryotic cell proliferation.

BACKGROUND AND SUMMARY OF THE INVENTION

Tissue culture allows the study in vitro of animal cell behavior in aninvestigator-controlled physiochemical environment. However, cellularmorphology and metabolic activity of cultured cells are affected by thecomposition of the substrate on which they are grown. Presumablycultured cells function best (i.e. proliferate and perform their naturalin vivo functions) when cultured on substrates that closely mimic theirnatural environment. Currently, studies in vitro of cellular functionare limited by the availability of cell growth substrates that presentthe appropriate physiological environment for proliferation anddevelopment of the cultured cells.

The ability of complex substrates to support cell growth in vitro hasbeen previously reported, and matrix products supporting such growth arecommercially available. For example, Becton Dickinson currently offerstwo such products: Human Extracellular Matrix and MATRIGEL® BasementMembrane Matrix. Human Extracellular Matrix is a chromatographicallypartially purified matrix extract derived from human placenta andcomprises laminin, collagen IV, and heparin sulfate proteoglycan.(Kleinman, HK et al., U.S. Pat. No. 4,829,000 (1989)) MATRIGEL® is asoluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS)tumor, gelled to form a reconstituted basement membrane. Both of thesematrix products require costly biochemical isolation, purification, andsynthesis techniques and thus production costs are high.

The present invention is directed to the use of vertebratesubmucosa-derived matrices as substrates for the growth and attachmentof a wide variety of cell types. The collagenous matrices for use inaccordance with the present invention comprise highly conservedcollagens, glycoproteins, proteoglycans, and glycosaminoglycans in theirnatural configuration and natural concentration. The extracellularcollagenous matrix for use in this invention is derived from submucosaltissue of a warm-blooded vertebrate. Submucosal tissue can be obtainedfrom various sources, including intestinal tissue harvested from animalsraised for meat production, including, for example, pigs, cattle andsheep or other warm-blooded vertebrates. This tissue can be used ineither its natural configuration or in a comminuted or partiallydigested fluidized form. Vertebrate submucosal tissue is a plentifulby-product of commercial meat production operations and is thus a lowcost cell growth substrate, especially when the submucosal tissue isused in its native layer sheet configuration.

The submucosal cell growth substrates of this invention provide cellswith a collagenous matrix environment in vitro resembling that found invivo. The natural composition and configuration of submucosal tissueprovides a unique cell growth substrate that promotes the attachment andproliferation of cells.

Accordingly, one object of the present invention is to provide arelatively inexpensive cell culture growth substrate that promotes orinduces growth and differentiation of cells cultured in vitro.

Another object of this invention is to provide a method for improvingcell proliferation in cell/tissue culture by using vertebrate submucosaltissue as a substrate for cell/tissue growth in vitro.

Another object of this invention is to provide a cell culturecomposition including a proliferating cell population in contact withsubmucosal tissue of a warm-blooded vertebrate and a nutrient medium forsupport of the growth of said cell population.

Still a further object of this invention is to provide a model systemfor studying tumor cell growth. The model system comprising aproliferating tumor cell population in contact with submucosal tissue ofa warm-blooded vertebrate and a nutrient medium.

It has been reported that compositions comprising submucosal tissue ofthe intestine of warm-blooded vertebrates can be used as tissue graftmaterials in sheet or fluidized form. U.S. Pat. No. 4,902,508 describestissue graft compositions that are characterized by excellent mechanicalproperties, including high compliance, a high burst pressure point, andan effective porosity index. These properties allow such compositions tobe used for vascular and connective tissue graft constructs. When usedin such applications the preferred graft constructs serve as a matrixfor the in vivo regrowth of the tissues replaced by the graftconstructs. U.S. Pat. No. 5,275,826 describes use of fluidized forms ofvertebrate submucosal tissues as injectable or implantable tissuegrafts.

An additional object of the present invention is to enhance or expandthe functional properties of vertebrate submucosal tissues as animplantable or injectable tissue graft construct by seeding thesubmucosal tissue in vitro with preselected or predetermined cell typesprior to implanting or injecting the graft construct into the host.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

There is provided in accordance with this invention a method andcomposition for supporting the proliferation and inducing tissuedifferentiation of eukaryotic cells cultured in vitro. Generally themethod comprises the step of contacting eukaryotic cells, in vitro, witha vertebrate submucosa-derived collagenous matrix under conditionsconducive to eukaryotic cell growth. The term "contacting" as usedherein with reference to cell culture is intended to include both directand indirect contact, for example in fluid communication, of thesubmucosal tissue and the cultured cells. The term "conditions conduciveto eukaryotic cell growth" as used herein refers to the environmentalconditions, such as sterile technique, temperature and nutrient supply,that are considered optimal for eukaryotic cell growth under currentlyavailable cell culture procedures. Although optimum cell cultureconditions used for culturing eukaryotic cells depend somewhat on theparticular cell type, cell growth conditions are generally well known inthe art. However a number of differentiated cell types are stillconsidered difficult to culture (i.e. islets of Langerhans, hepatocytes,chondrocytes, osteoblasts, etc.).

The collagenous matrix component of the present cell culture substrateis derived from vertebrate submucosa and comprises naturally associatedextracellular matrix proteins, glycoproteins and other factors.Preferably the collagenous matrix comprises intestinal submucosal tissueof a warm-blooded vertebrate. The small intestine of warm-bloodedvertebrates is a particularly preferred source of the cell culturesubstrate for use in this invention.

Suitable intestinal submucosal tissue typically comprises the tunicasubmucosa delaminated from the tunica muscularis and at least theluminal portion of the tunica mucosa. In one preferred embodiment of thepresent invention the intestinal submucosal tissue comprises the tunicasubmucosa and basilar portions of the tunica mucosa including the laminamuscularis mucosa and the stratum compactum which layers are known tovary in thickness and in definition dependent on the source vertebratespecies.

The preparation of submucosal tissue for use in accordance with thisinvention is described in U.S. Pat. No. 4,902,508, the disclosure ofwhich is expressly incorporated herein by reference. A segment ofvertebrate intestine, preferably harvested from porcine, ovine or bovinespecies, but not excluding other species, is subjected to abrasion usinga longitudinal wiping motion to remove the outer layers, comprisingsmooth muscle tissues, and the innermost layer, i.e., the luminalportion of the tunica mucosa. The submucosal tissue is rinsed withsaline and optionally sterilized; it can be stored in a hydrated ordehydrated state. Lyophilized or air dried submucosa tissue can berehydrated and used in accordance with this invention withoutsignificant loss of its cell proliferative activity.

The graft compositions of the present invention can be sterilized usingconventional sterilization techniques including glutaraldehyde tanning,formaldehyde tanning at acidic pH, propylene oxide treatment, gas plasmasterilization, gamma radiation, electron beam, peracetic acidsterilization. Sterilization techniques which do not adversely affectthe mechanical strength, structure, and biotropic properties of thesubmucosal tissue is preferred. For instance, strong gamma radiation maycause loss of strength of the sheets of submucosal tissue. Preferredsterilization techniques include exposing the graft to peracetic acid,1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of gammairradiation) or gas plasma sterilization; peracetic acid sterilizationis the most preferred sterilization method. Typically, the submucosaltissue is subjected to two or more sterilization processes. After thesubmucosal tissue is sterilized, for example by chemical treatment, thetissue may be wrapped in a plastic or foil wrap and sterilized againusing electron beam or gamma irradiation sterilization techniques.

The submucosal tissue specified for use in accordance with thisinvention can also be in a fluidized form. Submucosal tissue can befluidized by comminuting the tissue and optionally subjecting it toprotease digestion to form a homogenous solution. The preparation offluidized forms of submucosa tissue is described in U.S. Pat. No.5,275,826, the disclosure of which is expressly incorporated herein byreference. Fluidized forms of submucosal tissue are prepared bycomminuting submucosa tissue by tearing, cutting, grinding, or shearingthe harvested submucosal tissue. Thus pieces of submucosal tissue can becomminuted by shearing in a high speed blender, or by grinding thesubmucosa in a frozen or freeze-dried state to produce a powder that canthereafter be hydrated with water or a buffered saline to form asubmucosal fluid of liquid, gel or paste-like consistency. The fluidizedsubmucosa formulation can further be treated with a protease such astrypsin or pepsin at an acidic pH for a period of time sufficient tosolubilize all or a major portion of the submucosal tissue componentsand optionally filtered to provide a homogenous solution of partiallysolubilized submucosa.

The viscosity of fluidized submucosa for use in accordance with thisinvention can be manipulated by controlling the concentration of thesubmucosa component and the degree of hydration. The viscosity can beadjusted to a range of about 2 to about 300,000 cps at 25° C. Higherviscosity formulations, for example, gels, can be prepared from thesubmucosa digest solutions by adjusting the pH of such solutions toabout 6.0 to about 7.0.

Applicants have discovered that compositions comprising submucosaltissue can be used for supporting growth or proliferation of eukaryoticcells in vitro. Submucosal tissue can be used in accordance with thisinvention as a cell growth substrate in a variety of forms, includingits native sheet-like configuration, as a gel matrix, as an addition forart-recognized cell/tissue culture media, or as coating for culture-wareto provide a more physiologically relevant substrate that supports andenhances the proliferation of cells in contact with the submucosalmatrix. The submucosal tissue provides surfaces for cell adhesion andalso induces cell differentiation. The submucosal tissue is preferablysterilized prior to use in cell culture applications, however nonsterilesubmucosal tissue can be used if antibiotics are included in the cellculture system.

In one preferred embodiment cells are seeded directly onto sheets ofvertebrate submucosal tissue under conditions conducive to eukaryoticcell proliferation. The porous nature of submucosal tissue allowsdiffusion of cell nutrients throughout the submucosal matrix. Thus, forexample, cells can be cultured on either the luminal or abluminalsurface of the submucosal tissue. The luminal surface is the submucosalsurface facing the lumen of the organ source and typically adjacent toan inner mucosa layer in vivo whereas the abluminal surface is thesubmucosal surface facing away from the lumen of the organ and typicallyin contact with smooth muscle tissue in vivo.

Cells cultured on solid sheets of vertebrate submucosal tissue display adifferent growth pattern, and exhibit different interactions with thesubmucosal growth substrate, depending on which side of the submucosalsheet the cells are grown. Histological examination of tissue/cellscultured on intestinal submucosal tissue sheets in accordance with thisinvention reveals that cells that are seeded onto the abluminal surfacenot only grow/proliferate along the surface of the submucosal tissue,but they also more readily migrate into and proliferate within thesubmucosal tissue itself. The luminal surface comprises a more densematrix than the abluminal side and thus cells are less likely topenetrate the luminal side. Cells that are seeded onto the luminalsurface attach to the matrix but generally do not penetrate the surface.However certain cell types are capable of penetrating both the abluminaland luminal surfaces (eg squamous carcinoma cells and fibroblasts). Inaddition, certain cell types, such as fetal rat cells, when seeded onthe luminal side proliferate to form a polylayer of cells. Cells of thispolylayer can differentiate to perform functions characteristic of cellsin vivo and indicative of their position in the polylayer.

In one embodiment in accordance with the present invention thesubmucosal cell substrates can be utilized to provide a model system forstudying tumor cell growth. Tumor invasion of basement membranes is acrucial step in the complex multistage process which leads to theformation of metastases. Common features of the invasive processinclude: (a) attachment of the tumor cells to the basement membrane viacell surface receptors; (b) secretion of enzymes by the tumor cells thatcause degradation of the adjacent extracellular matrix (ECM) structures;and (c) migration of the cells through the matrix components. Tumorcells cultured in vitro however, typically form a monolayer or polylayerof flattened cells that is not appropriate for studying the process oftissue invasion by tumor cells.

Cell culture substrates comprising submucosal tissue of warm bloodedvertebrates can be used to culture various tumor cells types in vitro asa model of tumor cell growth characteristics. Such a model system wouldallow investigation of the molecular mechanisms involved in tumorinvasion and could ultimately lead to the development of novelanti-metastatic therapeutic strategies. In particular, such a modelsystem would enable the analysis in vitro of the effect of variouscompounds, such as growth factors, anti-tumor agents, chemotherapeutics,antibodies, irradiation or other factors known to effect cell growth, onthe growth characteristics of tumor cells.

To analyzing in vitro the effect of varying cell growth conditions onthe growth characteristics of tumor cells, the tumor cells are seeded ona cell growth substrate comprising submucosal tissue of a warm-bloodedvertebrate and provided a culture medium containing nutrients necessaryto the proliferation of said cells. The seeded cells are then culturedunder a preselected variable cell growth condition for varying lengthsof time and then the mucosal tissue substrate and the tumor cellpopulation on the substrate are histologically examined. The selectedgrowth condition can be the presence or concentration of a tumor cellgrowth modifier compound, such as cytokines or cytotoxic agents, in thenutrient medium. Alternatively the selected growth condition may be themodification of environmental factors such as temperature, pH,electromagnetic radiation, or nutrient composition. The effect of theselected growth condition on the morphology and growth of tumor cellscan then be assessed by histological analysis of control (tumor cellscultured in the absence of the selected growth condition) and test tumorcell cultures.

Both the fluidized and sheet forms of submucosal tissue can be used todevelop tumor cell invasion model systems. For example, the fluidizedform can be used to coat polycarbonate filters and then applied to aBoyden chamber-like device. Various stains, fluorescent markers, orradionucleotides can be used to obtain quantitative as well asqualitative invasion data. In addition, submucosal tissue can be used toassess the invasive potential of various cell types as well as a meansfor selective isolation of cells based upon their invasive potential.

In another embodiment of the present invention, cell growth substratesin accordance with the present invention are formed from fluidized formsof submucosal tissue. The fluidized submucosal tissue can be gelled toform a solid or semi-solid matrix. Eukaryotic cells can then be seededdirectly on the surface of the matrix and cultured under conditionsconducive to eukaryotic cell proliferation.

The cell growth substrate of the present invention can be combined withnutrients, including minerals, amino acids, sugars, peptides, proteins,or glycoproteins that facilitate cellular proliferation, such as lamininand fibronectin and growth factors such as epidermal growth factor,platelet-derived growth factor, transforming growth factor beta, orfibroblast growth factor. In one preferred embodiment fluidized orpowder forms of submucosal tissue can be used to supplement standardeukaryotic culture media to enhance the standard media's capacity forsustaining and inducing the proliferation of cells cultured in vitro.

In accordance with the present invention there is provided a cellculture composition for supporting growth in vitro of an eukaryotic cellpopulation in combination with submucosal tissue of a warm-bloodedvertebrate. The composition comprises nutrients, and optionally growthfactors required for optimal growth of the cultured cells. The submucosasubstrates of the present invention can be used with commerciallyavailable cell culture liquid media (both serum based and serum free).When grown in accordance with this invention, proliferating cells caneither be in direct contact with the submucosal tissue or they cansimply be in fluid communication with the submucosal tissue. It isanticipated that the cell growth compositions of the present inventioncan be used to stimulate proliferation of undifferentiated stems cellsas well as differentiated cells such as islets of Langerhans,hepatocytes and chondrocytes. Furthermore the described cell growthcomposition is believed to support the growth of differentiated cellswhile maintaining the differentiated state of such cells.

It has been well documented that submucosal tissue is capable ofinducing host tissue proliferation, remodeling and regeneration ofappropriate tissue structures upon implantation in a number ofmicroenvironments in vivo (e.g., tendon, ligament, bone, articularcartilage, artery, and vein). The use of such tissue in sheet form andfluidized forms for inducing the formation of endogenous tissues isdescribed and claimed in U.S. Pat. Nos. 5,281,422 and 5,275,826, thedisclosures of which are expressly incorporated by reference.

In one embodiment of the present invention the tissue replacementcapabilities of graft compositions comprising submucosal tissue ofwarm-blooded vertebrates are further enhanced or expanded by seeding thetissue with various cell types, prior to implantation. For example,submucosal tissue may be seeded with endothelial cells, keratinocytes,or islet of langerhans cells for use as a vascular graft, skinreplacement, or auxiliary pancreas, respectively. Alternatively, thesubmucosal tissue can be seeded with mesenchymal cells (stem cells)initially for expansion of the cell population and thereafter forimplantation into a host. Submucosal tissue can also serve as a deliveryvehicle for introducing genetically modified cells to a specificlocation in a host. The submucosal tissue for use in accordance withthis embodiment can either be in a fluidized form or in its native solidform. Optionally, after the submucosal tissue has been seeded witheukaryotic cells, the graft composition can be subjected to conditionsconducive to the proliferation of eukaryotic cells to further expand thepopulation of the seeded cells prior to implantation of the graft intothe host.

In one embodiment, compositions comprising submucosal tissue and aproliferating cell population can be encapsulated in a biocompatiblematrix for implantation into a host. The encapsulating matrix can beconfigured to allow the diffusion of nutrients to the encapsulated cellswhile allowing the products of the encapsulated cells to diffuse fromthe encapsulated cells to the host cells. Suitable biocompatiblepolymers for encapsulating living cells are known to those skilled inthe art. For example a polylysine/alginate encapsulation process hasbeen previously described by F. Lim and A. Sun (Science Vol. 210 pp.908-910). Indeed, vertebrate submucosa itself could be usedadvantageously to encapsulate a proliferating cell population on asubmucosal matrix in accordance with this invention for implantation asan artificial organ.

Submucosal tissue advantageously provides a physiological environmentthat supports the differentiation of cells cultured in vitro on thesubmucosal tissue. Thus, cell culture substrates comprising submucosaltissue can be used in combination with standard cell culture techniquesknown to those of ordinary skill in the art, to produce tissue grafts,in vitro, for implantation into a host in need thereof. The cells ofsuch a tissue perform their proper natural function based on cell typeand position within the submucosal tissue graft construct.

The method of forming a tissue graft in vitro comprises the steps ofseeding eukaryotic cells onto a cell growth substrate comprisingsubmucosal tissue of a warm-blooded vertebrate and culturing the cellsin vitro under conditions conducive to proliferation of the eukaryoticcells. Advantageously the synthesis in vitro of a tissue graftconstruct, wherein the cells of the tissue perform their proper naturalfunction, allows the generation of tissue grafts from an initially smallcell population that can be expanded in vitro prior to implantation.

EXAMPLE 1 Sterilization of Submucosal Tissue

Because cell culture techniques must be performed under strict asepticconditions, if antibiotics are not included in the culture system, thesubmucosa tissue must be prepared in a sterile manner for use as a cellculture substrate. Numerous sterilization methods have been investigatedto assess the effect of sterilization on the biotropic properties ofsubmucosal tissue. Sterilization techniques which do not significantlyweaken the mechanical strength and biotropic properties of the tissueare preferred. The following sterilization methods for intestinalsubmucosa have been evaluated: peracetic acid sterilization, 2.5 Mradgamma-irradiation, 1.0 Mrad gamma-irradiation, Exspor (Alcide, Norfolk,Conn.) sterilization and various combinations of these sterilizationmethods. Gamma-irradiation was performed on hydrated submucosal tissueusing a ⁶⁰ Cobalt-gamma chamber. Exspor sterilization was performedaccording to manufacturer's specifications using a sterilant volume (ml)to intestinal submucosa (g) ratio of 10 to 1.

Various cell types (e.g., IMR-90, FR, HT-29, RPEC) were seeded upon thesterilized submucosa and their growth characteristics were analyzed at1,3,7 and 14 days. Results obtained for all cell types showed thatsubmucosal derived growth substrates sterilized by gamma-irradiation orperacetic acid treatments supported some degree of adherence and growthof cells. However, cells seeded onto peracetic acid sterilizedsubmucosal derived substrates showed increased adherence, increasedsurvival, and enhanced rates of proliferation and differentiation;peracetic acid appears to be the preferred sterilization technique forpreparation of submucosa as a cell culture substrate.

EXAMPLE 2 Sterilization of Submucosal Tissue with Peracetic Acid

Submucosal tissue is soaked in a peracetic acid/ethanol solution for 2hours at room temperature using a ratio of 10:1 (mls peracetic solution:grams submucosal tissue) or greater. The peracetic acid/ethanol solutioncomprises 4% ethanol, 0.1% (volume: volume) peracetic acid and theremainder water. The 0.1% peracetic acid component is a dilution of a35% peracetic acid stock solution commercially available and defined asin table 1. Preferably, the submucosal tissue is shaken on a rotatorwhile soaking in the peracetic acid solution. After two hours, theperacetic acid solution is poured off and replaced with an equivalentamount of lactated Ringer's solution or phosphate buffered saline (PBS)and soaked (with shaking) for 15 minutes. The submucosal tissue issubjected to four more cycles of washing with lactated Ringer's or PBSand then rinsed with sterile water for an additional 15 minutes.

                  TABLE 1                                                         ______________________________________                                        Chemical Composition of the 35% Peracetic Acid Solution                       ______________________________________                                        Composition, % by weight                                                      Peracetic acid         35.5                                                   Hydrogen peroxide      6.8                                                    Acetic acid            39.3                                                   Sulfuric acid          1.0                                                    Water                  17.4                                                   Acetyl peroxide        0.0                                                    Stabilizer             500    PPM                                             Typical active oxygen analysis, % by weight                                   Active Oxygen as peracid                                                                             7.47                                                   Active Oxygen as H.sub.2 O.sub.2                                                                     2.40                                                   Total active oxygen    10.67                                                  ______________________________________                                    

EXAMPLE 3 Growth Characteristics Of Various Cell Types On SterilizedSubmucosa

Small intestinal submucosa was harvested and prepared from freshlyeuthanatized pigs as described in U.S. Pat. No. 4,902,508. Followingsterilization via various techniques (gamma irradiation, peracetic acid,etc.), the submucosal tissue was clamped within a polypropylene frame tocreate a flat surface area (50 mm²) for cell growth. The frame wassubmerged in culture medium to allow access of medium nutrients to bothsurfaces of the submucosal tissue. Various cell types were seeded (3×10⁴cells/submucosal tissue section) on the submucosal tissue and thenplaced in a 5% CO₂, 95% air incubator at 37° C. Following variousperiods of time, the seeded submucosal tissue was fixed in 10% neutralbuffered formalin, embedded in paraffin, and sectioned (6 um). Varioushistological and immunohistochemical staining procedures were thenapplied to determine the cell growth characteristics.

To date, the growth characteristics of the following cell lines havebeen studied using submucosal tissue as a growth substrate:

    ______________________________________                                        CELL LINE    CELL LINE DESCRIPTION                                            ______________________________________                                        CHO          Chinese hamster ovary cells                                      3T3          Swiss albino mouse embryo fibroblasts                            C3H10T1/2    C3H mouse embryo, multi-potential                                FR           Fetal rat skin (Sprague Dawley)                                  IMR90        Human fetal lung fibroblasts                                     HT-29        Human colon adenocarcinoma, moderately                                        well differentiated, grade II                                    RPEC         Rat pulmonary endothelial cells                                  HUVEC        Human umbilical vein cells                                       SCC-12       Squamous Cell Carcinoma                                          ______________________________________                                    

Table 2 summarizes various cell types and the corresponding specificmedium conditions used to culture on the submucosa derived cell culturesubstrates. The medium chosen represents optimal or near optimalconditions for propagation of each cell type under standard cell cultureconditions (i.e., plastic tissue culture flasks). All cell preparationswere incubated at 37° C. in a humidified atmosphere of 5% CO₂ /air.

                  TABLE 2                                                         ______________________________________                                        Cell types and corresponding culture conditions                               investigated using Intestinal Submucosal Tissue as a cell growth matrix       CELL TYPE        MEDIUM                                                       ______________________________________                                        3T3 (American Type Culture                                                                     DMEM (Dulbecco's modified                                    Collection (ATCC), CRL 1658)                                                                   Eagle's medium) with 1.5 g/L                                 Swiss mouse embryo fibroblasts                                                                 NaHCO.sub.3, 10% NNCS (neonatal                                               calf serum), 100 U/ml penicillin,                                             100 ug/ml streptomycin, 2 mM                                                  L-glutamine                                                  FR (ATCC, CRL 1213) cell line                                                                  DMEM, 10% NNCS, 100 U/ml                                     developed from a skin biopsy of a                                                              penicillin, 100 ug/ml                                        fetal (18 day gestation) germ-                                                                 streptomycin, 2 mM L-glutamine                               free Sprague Dawley rate                                                      HT-29 (ATCC, HTB 38) cell line                                                                 McCoy's, 10% NNCS, 100 U/ml                                  derived from human colon                                                                       penicillin, 100 ug/ml                                        adenocarcinoma   streptomycin, 2 mM L-glutamine                               HUV-EC-C (ATCC, CRL 1730)                                                                      F12 K medium, 10% FBS (fetal                                 endothelial cell line isolated                                                                 bovine serum), 100 ug/ml heparin,                            from human umbilical vein                                                                      50 ug/ml endothelial cell growth                                              supplement, 100 U/ml penicillin,                                              100 ug/ml streptomycin, 2 mM L-                                               glutamine                                                    IMR-90 (ATCC, CCL 186) human                                                                   McCoy's 5A medium, 20% NNCS,                                 diploid fibroblasts                                                                            100 U/ml penicillin, 100 ug/ml                                                streptomycin, 2 mM L-glutamine                               RPEC (J. P. Robinson, Purdue                                                                   RPMI 1640, 5% NCS (newborn calf                              University) endothelial cell line                                                              serum) 5% FBS (fetal bovine                                  derived from rat pulmonary                                                                     serum), 100 U/ml penicillin, 100                             endothelial cells                                                                              ug/ml streptomycin, 2 mM L-                                                   glutamine                                                    C3H10T1/2 (ATCC, CCL 226)                                                                      BME (basal medium Eagle), 10%                                mouse embryo fibroblasts                                                                       FBS, 100 U/ml penicillin, 100                                                 ug/ml streptomycin, 2 mM L-                                                   glutamin                                                     SCC-12 (W. Greenlee, Purdue                                                                    DMEM, 5% FBS (fetal bovine                                   University) squamous cell                                                                      serum), 4 mM L-glutamine, 1 mM                               carcinoma        sodium pyruvate                                              CHO (Chinese Hamster Ovary                                                                     F12 Medium 10% FBS with                                      Cells)           antibiotics (Neomycin)                                       ______________________________________                                    

The cellular growth on both the luminal and abluminal sides ofintestinal submucosal tissue has been investigated. Intestinalsubmucosal tissue as a growth substrate exhibits sidedness; that is, thecell/matrix interactions are different when the cells are cultured onthe abluminal versus the luminal side of the intestinal submucosaltissue. When selected cell types, such as rat FR cells are seeded on theluminal side, the cells attach to the matrix surface and proliferate toform a cellular polylayer. Alternatively, when FR cells are seeded onthe abluminal side, the cells not only grow along the surface but alsomigrate into the submucosal matrix.

The stratum compactum of the luminal side of vertebrate intestinalsubmucosal tissue provides a dense connective tissue matrix and morereadily supports monolayer or polylayer formation of select cell types(i.e. endothelial and epithelial cells). Alternatively, the abluminalside represents a more loose connective tissue structure that morereadily supports migration of cells within the matrix structure (i.e.fibroblasts).

IMR-90 fibroblasts, when seeded upon the abluminal or luminal sides ofthe intestinal submucosal tissue, quickly became adherent andproliferated throughout the matrix components. These cells illustratedtheir characteristic spindle shape and large vesicular nucleus withinthe extracellular matrix components. However, 3T3 fibroblasts showedminimal adherence and growth potential when seeded upon the intestinalsubmucosal tissue.

Endothelial cells formed a confluent monolayer of spindle-shaped cellsalong the stratum compactum surface of the intestinal submucosal tissuewithin 3 days. At later times the monolayer became more dense and somecells intercalated down into the matrix components. Interestingly, someendothelial cells that penetrated into the matrix components formed alining along the lumen of structures representing original blood vesselsof the native intestine.

To date, the growth characteristics of the following primary cellstrains have been studied using intestinal submucosal tissue as a growthsubstrate:

CELL STRAIN

Rat Cardiac Muscle

Porcine Smooth Muscle (aorta)

Porcine Endothelial (aorta)

Rabbit Smooth Muscle (aorta)

Rabbit Endothelial(aorta)

Porcine Smooth Muscle and Endothelial (mixed & co-cultured)

Human Osteoblasts

Human Endothelial Cells

Primary cell strains are cells that have been harvested from an organismand placed in culture. Subsequent passages of these cells (from 2-3times) using standard in vitro cell culture techniques (to increase thenumber of cells) were frozen for later use. Each of the above listedcell strains was thawed, cultured in the presence of intestinalsubmucosal tissue and examined histologically. Each of the cultured cellstrain populations proliferated and retained their differentiatedappearance as determined by histological examination. For example, after7-14 days of culture on intestinal submucosal tissue: the humanosteoblast cells continued to accumulate appatite crystals and respondto osteogenic stimuli such as hormones; rat cardiac muscle cellsretained their contractile properties; porcine smooth muscle cellsretained smooth muscle actin; and porcine endothelial cells made factoreight.

EXAMPLE 4 Intestinal Submucosal Cell Culture Substrates as a Tumor CellGrowth Model System

The morphology and invasive properties of an established cell line froma human squamous cell carcinoma of the face known as SCC-12 (obtainedfrom W. Greenlee, Purdue University) cultured in vitro were studied.When grown under standard cell culture conditions for skin cells (e.g.,gamma-irradiated or mitomycin C-treated feeder layer of Swiss 3T3 mousefibroblasts) a monolayer of flattened cells is formed. However SCC-12cells when seeded upon the abluminal surface of intestinal submucosaltissue, showed, upon histological examination active degradation of thesubmucosal matrix components and invasion of the intestinal submucosaltissue.

SCC-12 cells were seeded (3×10⁴ cells/0.8 cm² of intestinal submucosaltissue) on either the abluminal or luminal surface of sterilizedintestinal submucosal tissue and floated in growth medium consisting ofDMEM containing 5% fetal calf serum, 4 mM L-glutamine, and 1 mM sodiumpyruvate. At timepoints representing 3, 7, 14, and 21 days, the growthcharacteristics were analyzed using standard histologic techniques. Onday 3, the cells were strongly adherent and appeared to form acontinuous layer (1-2 cells thick) along surface of the intestinalsubmucosal tissue. Morphologically, the cells were round and activelyproducing extracellular matrix products. After 7 days, a significantdifference was noted in the cells ability to invade the abluminal versusthe luminal surface of the intestinal submucosal tissue. The layer ofcells along the luminal surface of the intestinal submucosal tissueappeared to only increase in density. Alternatively, those cells seededupon the abluminal surface, showed active degradation of the submucosalmatrix components and penetration up to 30 um. At longer durations,there was an increasing number of cells at greater depths of penetrationand a greater extent of intestinal submucosal tissue degradation.Although the SCC-12 cells actively invade intestinal submucosal tissuefrom both the abluminal and luminal surfaces, the observed invasion ratewas greater when SCC-12 cells were placed on the abluminal side.

EXAMPLE 5 Intestinal Submucosal Tissue Supports Cytodifferentiation

FR Epithelial cells form a stratified polylayer when cultured on theluminal (stratum compactum) side of intestinal submucosal tissue. Cellsadjacent to the intestinal submucosal tissue were columnar in shape andbecame progressively more flattened near the surface of the polylayer.After 14 days, structures resembling desmosomes were identified and thecellular layer stained positively for cytokeratin with a pan cytokeratinantibody. In addition, it appeared that the epithelial cells producedsupporting matrix products (potentially basement membrane) as they do invivo under normal healthy conditions. These findings suggest that theintestinal submucosal tissue supports natural epithelial cell maturationand differentiation processes.

The observed stratification of FR cells grown on the luminal side(stratum compactum) of a submucosal growth substrate provides evidencethat the intestinal submucosal tissue supports and induces cellulardifferentiation in vitro. To verify the induction of cytodifferentiationof the FR cells, immunohistochemical and immunofluorescence analyseswere performed for detecting the production of cytokeratin by FR cellscultured in the presence and absence of intestinal submucosal tissue.Cytokeratin is a predominant intracellular structural protein producedby terminally differentiated epithelial cells known as keratinocytes.Immunohistochemistry was performed on the protease-digested,formalin-fixed, paraffin embedded sections of FR cells grown onintestinal submucosal tissue using an anti-pan cytokeratin (C2931,Sigma, St. Louis, Mo.) as the primary antibody. Immunodetection wasperformed using the avidin-biotin complex (ABC) method and the Biogenexsupersensitive StriAviGen kit (Vector Laboratories, Burlingame, Calif.).Tissue sections representing rat skin biopsies and HT29 cells grown onintestinal submucosal tissue were included in the analysis as positiveand negative controls, respectively.

Results indicated a gradation of cytokeratin staining along the FRcellular polylayer with those cells at the surface of the polylayerstaining most intensely. A similar positive staining pattern wasobserved in the cells forming the epidermal layer of the rat skin.However, no cytokeratin was detected in the specimens representing HT29cells cultured on intestinal submucosal tissue.

An immunofluorescence analysis for cytokeratin was performed using flowcytometry to determine if the FR cell line expressed the differentiationproduct cytokeratin under standard culture conditions (in the absence ofintestinal submucosal tissue). Swiss 3T3 Fibroblast (3T3) and squamouscell carcinoma (SCC-12) cell lines were included in the analysis asnegative and positive controls respectively. Cells were harvested fromtissue culture flasks, permeabilized using a cold methanol pretreatment,and incubated in the presences of anti-pan cytokeratin antibody atvarious dilutions (including the absence of anti-pan cytokeritinantibody to serve as a control). A goat anti-mouse antibody conjugatedwith fluorescein isothiocyanate (GAM-FITC) was then applied tofacilitate immunodetection. The cell preparations were then analyzed ona EPICS Elite flow cytometer (Coulter Corp., Hialeah, Fla.) using 488 nmexcitation produced by an air-cooled argon laser. Fluorescence emissionswere measured at 525 nm with a bandpass filter. Untreated cells andcells treated only with GAM-FITC were also analyzed to establishbackground fluorescence levels. Table 3 represents the relativepercentage of FITC fluorescence for each cell type following indirectimmunofluorescence staining. As the data indicate only the positivecontrol SCC-12 cell line expresses cytokeratin and the FR cell line doesnot express cytokeratin under standard culture conditions in the absenceof submucosal substrate.

                  TABLE 3                                                         ______________________________________                                        Indirect Immunofluorescence Analysis for                                      Cytokeratin BCC-12, 3T3 and FR Cells                                          Cell       Dilution of Anti-                                                                         Percent GAM-FITC                                       Type       Pan Cytokeratin                                                                           Fluorescence                                           ______________________________________                                        SCC-12     0 (control)  2%                                                    SCC-12     1:100       72%                                                    SCC-12     1:400       74%                                                    SCC-12     1:1000      76%                                                    SCC-12     1:4000      72%                                                    3T3        0 (control) 11%                                                    3T3        1:100       10%                                                    3T3        1:400       18%                                                    3T3        1:1000       8%                                                    3T3        1:4000       5%                                                    FR         0 (control)  6%                                                    FR         1:100       11%                                                    FR         1:400        6%                                                    FR         1:1000       4%                                                    FR         1:4000       4%                                                    ______________________________________                                    

EXAMPLE 6 Isolation Of Hamster Pancreatic Islets

Hamster pancreatic islets were isolated as previously described by Gotohet al. (Transportation Vol. 43, pp. 725-730(1987)). Briefly, 6-8 weekold Golden hamsters (Harlan, Indianapolis, Ind.) were anesthetized viainhalation of Metofane (Methoxyflurane; Pitman-Moore; Mundelein, Ill.).The common bile duct was cannulated under a stereomicroscope with apolyethylene catheter (PE-10 tubing; CMS; Houston, Tex.), through whichapproximately 3-4 mls of ice cold M-199 medium (commercially availablefrom Gibco BRL) containing 0.7 mg/ml of collagenase P was injectedslowly until whole pancreas was swollen. The pancreas was excised anddigested at 37° C. for approximately 50 minutes in M-199 mediumcontaining 100 μg/ml of penicillin G and 100 μg/ml of streptomycin (noadditional collagenase). The digest was washed three times in ice coldM-199 medium and passed sequentially through a sterile 500 μm stainlesssteel mesh, then a 100 μm mesh. Following purification by centrifugationthrough a ficoll density gradient (1.045, 1.075, 1.085 and 1.100) at 800g for 10 min, islets were recovered from the top two interfaces.

Culturing of Pancreatic Islet Cells on Intestinal Submucosal Tissue

Islets of Langerhans (islet cells) were cultured on submucosal cellgrowth substrates at 37° C. in an incubator supplemented with 5% CO and95% air. The islet cells were cultured in the presence of various formsof intestinal submucosal tissue using the following procedures:

1. Direct Contact: Intestinal submucosal tissue and the cultured cellsphysically contact one another.

2. Indirect Contact: Intestinal submucosal tissue and the cultured cellsare separated by a stainless steel mesh.

3. Solubilized intestinal submucosal tissue is added to the culturemedia

4. Cells are cultured on solubilized intestinal submucosa coated cultureplate. The coating was applied by placing 1 ml of solubilized intestinalsubmucosal tissue in a 35 mm culture plate, heated at 37° C. for 2hours, removing the excess intestinal submucosal tissue fluid byaspiration and washing the coated plates once with culture media.

In direct contact culture method, an intestinal submucosa membrane ofapproximately 1×1 cm was placed on top of stainless steel mesh with thestratum compactum side facing up. Isolated islets were then placed ontothe membrane and continuously cultured in M-199 medium (commerciallyavailable from Gibco BRL) for 7 days. Cell proliferation was examinedevery second day under a stereomicroscope and was compared with thecontrol group (cultured in the absence of submucosa tissue).

Sterilization of Submucosal Tissue Before Co-culturing

1. Intestinal submucosal tissue derived cell culture substrates weresterilized by several different means: peracetic acid treatment or gammairradiation. Gamma irradiated and the native (no further treatment afterisolation of the intestinal submucosal tissue) membranes can be useddirectly as cell culture substrates provided they have been sufficientlyrehydrated with the culture media prior to the co-culture (nativemembranes must be cultured in the presence of antibiotics). Peraceticacid sterilized membranes, must first be washed to remove residualperacetic acid prior to culturing since peracetic acid residue may betoxic to the cells. Typically peracetic acid sterilized tissues weresoaked in a large quality of medium for 24 hours followed by extensivewashing with the same medium.

2. Solubilized forms of intestinal submucosal tissue were sterilized bydialyzing against 6.5% chloroform in either 0.1M acetic acid(AA-submucosa) or phosphate buffered saline (PBS-submucosa) for 2 hoursat room temperature. The exterior surface of the dialysis tubing issterilized by rinsing the outside of the tubing with 70% alcohol priorto removal of the intestinal submucosal tissue. The dialysis tubing hasa molecular weight cut-off of 12,000-14,000; thus, proteins retainedinside tubing are those with molecular weight greater than 14,000.

Results

In the control group (islets cultured in the absence of submucosatissue) examination of seven day cultures revealed that fibroblast cellshad overgrown the islet cells.

When islet cells were cultured on growth substrates comprisingintestinal submucosal tissue, overgrowth of the islet cells byfibroblast cells did not occur. In intestinal submucosal tissue directculture systems, the islets became loosely packed with many cellssurrounding the islet capsule. Cells migrated from the capsule and cellproliferation occurred on top of the membrane in the absence offibroblast overgrowth. Culturing islet cells on intestinal submucosaltissue coated culture ware also appeared to facilitate migration ofepithelioid cells out of the islet capsule. Further attachment to thecoating surface and the formation of a monolayer of epithelioid cellswas observed.

These data indicate that submucosal substrates can be used to stimulategrowth of islet cells in vitro without overgrowth of fibroblast cells.Islet cells can thus be isolated from pancreatic tissue and grown invitro in contact with a cell growth substrate comprising intestinalsubmucosal tissue of a warm-blooded vertebrate under conditionsconducive to the proliferation of the islet cells and without concurrentgrowth of fibroblasts. These islet cell culture compositions remainsubstantially free of fibroblast overgrowth.

We claim:
 1. A method for enhancing the capabilities of a submucosaltissue graft construct to repair damaged or diseased tissue, said methodcomprising the step of seeding the submucosal tissue with cells selectedfrom the group consisting of endothelial cells, keratinocytes andmesenchymal cells prior to implanting or injecting the graft constructinto a host.
 2. The method of claim 1 further comprising the step ofsubjecting the seeded graft construct to conditions conducive to theproliferation of the cells prior to implanting or injecting the graftmaterial into the host.
 3. The method of claim 1 wherein the submucosaltissue comprises tunica submucosa delaminated from both the tunicamuscularis and at least the luminal portion of the tunica mucosa ofvertebrate intestinal tissue.
 4. The method of claim 1 wherein the cellshave been genetically modified.
 5. The method of claim 1, wherein thesubmucosal tissue is fluidized submucosal tissue.
 6. The method of claim5, wherein the fluidized submucosal tissue comprises submucosal tissuedigested with an enzyme for a period of time sufficient to solubilizethe tissue.